Abstract

Improving biomass yield is a major goal of Miscanthus breeding. We conducted a study on one interspecific Miscanthus sinensis x Miscanthus sacchariflorus F 1 population and two intraspecific M. sinensis F 1 populations, each of which shared a common parent. A field trial was established at Urbana, IL during spring 2011, and phenotypic data were collected in 2012 and 2013 for fourteen yield traits. Six high-density parental genetic maps, as well as a consensus genetic map integrating M. sinensis and M. sacchariflorus, were developed via the pseudotestcross strategy for noninbred parents with ≥1214 single-nucleotide polymorphism markers generated from restriction site-associated DNA sequencing. We confirmed for the first time a whole-genome duplication in M. sacchariflorus relative to Sorghum bicolor, similar to that observed previously for M. sinensis. Four quantitative trait locus (QTL) analysis methods for detecting marker-trait associations were compared: (1) individual parental map composite interval mapping analysis, (2) individual parental map stepwise analysis, (3) consensus map single-population stepwise analysis and (4) consensus map joint-population stepwise analysis. These four methods detected 288, 264, 133 and 109 total QTLs, which resolved into 157, 136, 106 and 86 meta-QTLs based on QTL congruency, respectively, including a set of 59 meta-QTLs common to allmore » four analysis methods. Composite interval mapping and stepwise analysis co-identified 118 meta-QTLs across six parental maps, suggesting high reliability of stepwise regression in QTL detection. Joint-population stepwise analysis yielded the highest resolu-tion of QTLs compared to the other three methods across all meta-QTLs. Strong, frequently advantageous trans-gressive segregation in the three populations indicated a promising future for breeding new higher-yielding cultivars of Miscanthus.« less

@article{osti_1375469,
title = {Genetic mapping of biomass yield in three interconnected Miscanthus populations},
author = {Dong, Hongxu and Liu, Siyao and Clark, Lindsay V. and Sharma, Shailendra and Gifford, Justin M. and Juvik, John A. and Lipka, Alexander E. and Sacks, Erik J.},
abstractNote = {Improving biomass yield is a major goal of Miscanthus breeding. We conducted a study on one interspecific Miscanthus sinensis x Miscanthus sacchariflorus F1 population and two intraspecific M. sinensis F1 populations, each of which shared a common parent. A field trial was established at Urbana, IL during spring 2011, and phenotypic data were collected in 2012 and 2013 for fourteen yield traits. Six high-density parental genetic maps, as well as a consensus genetic map integrating M. sinensis and M. sacchariflorus, were developed via the pseudotestcross strategy for noninbred parents with ≥1214 single-nucleotide polymorphism markers generated from restriction site-associated DNA sequencing. We confirmed for the first time a whole-genome duplication in M. sacchariflorus relative to Sorghum bicolor, similar to that observed previously for M. sinensis. Four quantitative trait locus (QTL) analysis methods for detecting marker-trait associations were compared: (1) individual parental map composite interval mapping analysis, (2) individual parental map stepwise analysis, (3) consensus map single-population stepwise analysis and (4) consensus map joint-population stepwise analysis. These four methods detected 288, 264, 133 and 109 total QTLs, which resolved into 157, 136, 106 and 86 meta-QTLs based on QTL congruency, respectively, including a set of 59 meta-QTLs common to all four analysis methods. Composite interval mapping and stepwise analysis co-identified 118 meta-QTLs across six parental maps, suggesting high reliability of stepwise regression in QTL detection. Joint-population stepwise analysis yielded the highest resolu-tion of QTLs compared to the other three methods across all meta-QTLs. Strong, frequently advantageous trans-gressive segregation in the three populations indicated a promising future for breeding new higher-yielding cultivars of Miscanthus.},
doi = {10.1111/gcbb.12472},
journal = {Global Change Biology. Bioenergy},
number = ,
volume = ,
place = {United Kingdom},
year = {2017},
month = {7}
}

Miscanthus is especially attractive as a bioenergy crop for temperate environments because it produces high yields, needs few inputs, and grows well during the cool weather of early spring and late fall when few warm-season grasses can. However, Miscanthus feedstock production for the emerging U.S. bioenergy industry and for existing demand in Europe is based on a single sterile, vegetatively propagated variety of M. ×giganteus. M. ×giganteus is an interspecific hybrid of the parental species M. sinensis and M. sacchariflorus. Prior to the current study, little information existed about the genetic diversity and breeding potential of either M. ×giganteus parentalmore » species. In the current project, we studied more than 600 accessions of M. sinensis from throughout its native range in China, Japan, and Korea, in addition to ornamental cultivars and U.S. naturalized populations. Using thousands of DNA markers, we identified seven geographically distinct genetic groups of M. sinensis. Notably, we found that the ornamental cultivars and U.S. naturalized populations were derived from only a subset of the Southern Japan group, indicating that our study greatly increased the genetic diversity available for breeding new biomass cultivars. Additionally, this new understanding of M. sinensis population structure could be used to predict which crosses may produce progeny with the greatest hybrid vigor. Replicated field trials were also established at multiple locations in North America and Asia. Data on traits of importance for biomass productivity, such as flowering time, yield and height, were taken. Analyses of the phenotypic data from the field trials along with the DNA markers allowed us to identify many marker-trait associations. These results will enable marker-assisted breeding, which will allow selection at the seedling stage rather than waiting two to three years to obtain phenotypic data. Thus, this study is expected to greatly increase the efficiency of breeding Miscanthus for improved adaptation and biomass yield.« less

Miscanthus is a perennial C4 grass that has recently become an important bioenergy crop. The efficiency of breeding improved Miscanthus biomass cultivars could be greatly increased by marker-assisted selection. Thus, a high-density genetic map is critical to Miscanthus improvement. In this study, a mapping population of 261 F1 progeny was developed from a cross between two diploid M. sinensis cultivars, ‘Strictus’ and ‘Kaskade’. High-density genetic maps for the two parents were produced with 3044 newly developed single nucleotide polymorphisms (SNPs) obtained from restriction site-associated DNA sequencing, and 138 previously mapped GoldenGate SNPs. The female parent (‘Strictus’) map spanned 1599 cM,more » with 1989 SNPs on 19 linkage groups, and an average intermarker spacing of 0.8 cM. The length of the male parent (‘Kaskade’) map was 1612 cM, with 1821 SNPs, and an average intermarker spacing of 0.9 cM. The utility of the map was confirmed by locating quantitative trait loci (QTL) for the zebra-striped trait, which was segregating in this population. Three QTL for zebra-striped presence/absence (zb1, zb2 on LG 7, and zb3 on LG 10) and three for zebra-striped intensity (zbi1, zbi2, zbi3 on LGs 7, 10, 3) were identified. Each allele that caused striping was recessive. Incomplete penetrance was observed for each zb QTL, but penetrance was greatest when two or more zb QTL were homozygous for the causative alleles. Similarly, the intensity of striping was greatest when two or more zbi QTL were homozygous for alleles that conferred the trait. Comparative mapping indicated putative correspondence between zb3 and/or zbi2 on LG 10 to previously sequenced genes conferring zebra stripe in maize and rice. These results demonstrate that the new map is useful for identifying marker–trait associations. The mapped markers will become a valuable community resource, facilitating comparisons among studies and the breeding of Miscanthus.« less

Specific Objectives: 1. To identify the gene(s) underlying a major QTL for stem sugar concentration located on chromosome 3. 2. To identify QTL for stem juice volume and stalk sugar concentration and to identify the underlying genes. 3. To classify 60 novel sorghum bmr mutants from the USDA TILLING population in allelic groups based on cell wall chemistry and allelism tests. 4. To select representative bmr mutants from each allelic group and selected NIR spectral mutants for their potential value as feedstock for ethanol production. 5. To clone and characterize those Bmr genes that represent loci other than Bmr12 andmore » Bmr6 using a mapping and a candidate gene approach. Objective 1 The experiments for this objective are largely complete and the data have been analyzed. Data interpretation and follow-up experiments are still in progress. A manuscript is in preparation (Vermerris et al.; see publication list for full details). The main results are: 1) 16 cDNA libraries were prepared and sequenced at Cornell University. The libraries represent internode tissue and flag leaf tissue at booting, internode tissue and peduncle at soft-dough stage, from two plants per sampling time with the Rio allele for the QTL on chromosome 3, and two plants with the BTx623 allele on chromosome 3 (4 tissues x 2 genotypes x 2 replicates) 2) 480 million 86-nucleotide reads were generated from four lanes of Illuminia HiSeqII 3) 74% of the reads could be mapped to the sorghum transcriptome, indicative of good sequence quality 4) Of the 216 genes within the QTL, 17 genes were differentially expressed among plants with and without the Rio QTL. None of these 17 genes had obvious roles in sucrose metabolism 5) Clustering algorithms identified a group of 721 co-expressed genes. One of these genes is a sucrose synthase gene. This cluster also contains 10 genes from the QTL. 6) Among these co-expressed genes are regulatory genes for which knock-out lines in Arabidopsis have been obtained. Analysis of these lines is in progress. Objective 2 The experiments from this objective have been completed and the data were published in the journal Crop Science by Felderhoff et al. (2012). A second publication by Felderhoff et al. is in progress (see publication list for full details). The experiments were based on a mapping population derived from the sweet sorghum 'Rio' and the dry-stalk grain sorghum BTx3197. The main findings were: 1) The apparent juiciness of the sorghum stalk, based on the appearance of a cut stem surface (moist vs. pithy), is not representative of the moisture content of the stalk. This was surprising, as pithy stalks have been associated with low moisture content. This means that in order to assess 'juiciness', a different evaluation needs to be used, for example by removing juice with a roller press or by measuring the difference in mass between a fresh and dried stalk segment. 2) A total of five QTLs associated with juice volume (corrected for height) or moisture content were identified, but not all QTLs were detected in all environments, providing evidence for genotype x environment interactions. This finding complicates breeding for juice volume using marker-assisted selection. 3) The QTL for sugar concentration identified on chromosome 3, and the subject of Objective 1, was confirmed in this mapping population, but unlike in previous studies (Murray et al., 2008), the presence of this QTL was associated with negative impacts on agronomic performance (fresh and dry biomass yield, juice yield). Consequently, introgression of the Brix QTL from Rio as part of a commercial breeding program will require monitoring of the precise impacts of this QTL on agronomic performance. 4) The absence of dominance effects for the Brix trait (= sugar concentration) indicated that Brix must be high in both parents to produce high Brix in hybrids. This means an extra constraint on the development of hybrid parents. With the results from Objective 1, the selection of progeny containing favorable alleles for sugar concentration is expected to be more efficient. Objectives 3 and 4 The experiments from these objectives have been completed. Some of the data have been published in the journal BioEnergy Research (Sattler et al. 2012) and in a book chapter on the utilization of sorghum biomass by Vermerris and Saballos (2012) (see publication list for full details). One manuscript is in progress and is expected to be submitted in 2013. The experiments for these objectives were based on the characterization of a set of novel sorghum mutants identified in a TILLING population generated by Dr. Zhanguo Xin (USDA-RS, Lubbock, TX). The main findings were: 1) Based on allelism tests of bmr mutants from the USDA TILLING population, there are three novel sorghum bmr loci, currently referred to as bmr-20, bmr-100 and bmr-1107. This brings the total number of bmr loci to a maximum of seven (manuscript in preparation). 2) The biomass conversion properties of the novel bmr mutants are not significantly better than the wildtype control, limiting their utility for bioenergy production (manuscript in preparation). This also means that all three bmr loci that positively influence biomass conversion (bmr2, bmr6 and bmr12) have been cloned. Two of these genes (Bmr6 and Bmr2) were cloned with funding from this project. 3) Four novel mutant alleles of bmr12 were identified and characterized. These mutants alleles are bmr12-30, bmr12-34, bmr12-35and bmr12-820, and they all contain missense mutations, leading to amino acid substitutions with varying effects on lignin content and lignin subunit composition (syringyl/guaiacyl ratio). One of the mutants, bmr-35, represents a phenotype that is intermediate between the wild type and the bmr12-reference mutant, which is a null mutant. This intermediate phenotype may offer a balance between enhanced biomass conversion properties and good agronomic performance (Sattler et al., 2012). 4) It is possible to identify sorghum mutants with altered biomass conversion properties using analysis of leaf segments by near infrared reflectance spectroscopy (NIRS). Approximately 10% of 200 M3 families contained spectral outliers suggestive of cell wall changes, and half of those showed variation in biomass conversion efficiency (Vermerris and Saballos, 2012). Objective 5 The experiments from this objective were completed and the data were published by Saballos et al. (2012) in The Plant Journal. The main findings were: 1) The Bmr2 gene encodes the main 4-coumarate CoA ligase in sorghum; the genetic proof consisted of showing how two independent mutations in this gene both resulted in the same phenotype, and by showing that these mutations were allelic. Reduced Bmr2 activity leads to reduced lignin content and brown vascular tissue. 2) Allele-specific molecular markers were developed so that the inheritance of these recessive alleles can be tracked in sorghum breeding programs aimed at improving biomass conversion. 3) Together with four other bona fide 4CL genes, the Bmr2 gene is a member of a multigene family in sorghum. Based on phylogentic analysis, one of those genes is involved in flavonoid metabolism, the others in monolignol biosynthesis. Enzymatic activities for the enzymes encoded by Bmr2 and itsparalogs were determined. 4) Both bmr2 mutations are missense mutations that result in the substitution of apolar amino acids with polar amino acids. In both cases, these substitutions are in hydrophobic domains, which destabilize the protein, leading to degradation. This is apparent from western blots and activity assays with heterologously expressed enzymes. 5) The plant tries to compensate for the reduced 4CL activity by increasing the expression of Bmr2 and its paralogs. As a result of the higher expression levels of the paralogs, there is enough 4CL activity to minimize negative impacts on growth and development. List of all publications to date in which the funding of this project is acknowledged 1) Vermerris W, Saballos A (2012) Genetic enhancement of sorghum for biomass utilization. In Paterson, A. (Ed.) Genetics and Genomics of the Saccharinae, Springer, New York, NY. pp. 391-428. 2) Felderhoff T, Murray SC, Klein PE, Sharma A, Hamblin MT, Kresovich S, Vermerris W, Rooney, WL (2012) QTLs for energy-related traits in a sweet x grain sorghum [Sorghum bicolor (L.) Moench] mapping population. Crop Science 52: 2040-2049. 3) Sattler SE, Palmer NA, Saballos A, Greene AM, Xin Z, Sarath G, Vermerris W, Pedersen JF (2012) Identification and characterization of four missense mutations in Brown midrib12 (Bmr12), the caffeic acid O-methyltranferase (COMT) of sorghum. BioEnergy Research (in press) DOI 10.1007/s12155-012-9197-z 4) Saballos A, Sattler S, Sanchez E, Foster TP, Xin Z, Kang CH, Pedersen J, Vermerris W (2012). Brown midrib2 encodes the major 4-coumarate:CoA ligase involved in lignin biosynthesis in sorghum (Sorghum bicolor (L.) Moench). The Plant Journal 70: 818-830. doi: 10.1111/j.1365-313X.2012.04933. 5) Vermerris, W (2011) Survey of genomics approaches to improve bioenergy traits in maize, sorghum and sugarcane. Journal of Integrative Plant Biology 53: 105-119 6) Saballos A, Ejeta G, Sanchez E, Kang CH, Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L.) Moench] identifies SbCAD2 as the Brown midrib6 gene. Genetics 181: 783-795. 7) Saballos A, Vermerris W, Rivera L, Ejeta G (2008) Allelic association, chemical characterization and saccharification properties of brown midrib mutants of sorghum (Sorghum bicolor (L.) Moench). BioEnergy Research 2: 193-204 8) Felderhoff TJ. (2012) QTLs for energy related traits in a sweet x grain RIL sorghum [Sorghum bicolor (L.) Moench] population. M.S. Thesis, Texas A&M University. Publications in preparation (tentative titles) 9) Felderhoff T, Murray SC, Klein PE, Sharma A, Hamblin MT, Kresovich S, Vermerris W, Rooney, WL (2013) QTLs for biomass and juice composition in a sweet x grain sorghum [Sorghum bicolor (L.) Moench] mapping population. 10) Vermerris W, Fear J, Saballos A, Murray SC, Rooney WL, Kresovich S. Identification of candidate genes for sucrose accumulation in sweet sorghum using RNA-seq. 11) Sattler SE, Palmer NA, Saballos A, Xin Z, Vermerris W, Pedersen JF. Characterization of novel sorghum brown midrib mutants. Presentations since last progress report truncated due to space limitations.« less

Abstract Background and Aims Miscanthus, a C4 perennial grass native to East Asia, is a promising biomass crop. Miscanthus sacchariflorus has a broad geographic range, is used to produce paper in China and is one of the parents (along with Miscanthus sinensis) of the important biomass species Miscanthus × giganteus. The largest study of M. sacchariflorus population genetics to date is reported here. Methods Collections included 764 individuals across East Asia. Samples were genotyped with 34 605 single nucleotide polymorphisms (SNPs) derived from restriction site-associated DNA sequencing (RAD-seq) and ten plastid microsatellites, and were subjected to ploidy analysis by flowmore » cytometry. Key Results Six major genetic groups within M. sacchariflorus were identified using SNP data: three diploid groups, comprising Yangtze (M. sacchariflorus ssp. lutarioriparius), N China and Korea/NE China/Russia; and three tetraploid groups, comprising N China/Korea/Russia, S Japan and N Japan. Miscanthus sacchariflorus ssp. lutarioriparius was derived from the N China group, with a substantial bottleneck. Japanese and mainland tetraploids originated from independent polyploidization events. Hybrids between diploid M. sacchariflorus and M. sinensis were identified in Korea, but without introgression into either parent species. In contrast, tetraploid M. sacchariflorus in southern Japan and Korea exhibited substantial hybridization and introgression with local diploid M. sinensis. Conclusions Genetic data indicated that the land now under the Yellow Sea was a centre of diversity for M. sacchariflorus during the last glacial maximum, followed by a series of migrations as the climate became warmer and wetter. Overall, M. sacchariflorus has greater genetic diversity than M. sinensis, suggesting that breeding and selection within M. sacchariflorus will be important for the development of improved M. × giganteus. Ornamental M. sacchariflorus genotypes in Europe and North America represent a very narrow portion of the species’ genetic diversity, and thus do not well represent the species as a whole.« less

Abstract Background and AimsMiscanthus, a C4 perennial grass native to East Asia, is a promising biomass crop. Miscanthus sacchariflorus has a broad geographic range, is used to produce paper in China and is one of the parents (along with Miscanthus sinensis) of the important biomass species Miscanthus × giganteus. The largest study of M. sacchariflorus population genetics to date is reported here. MethodsCollections included 764 individuals across East Asia. Samples were genotyped with 34 605 single nucleotide polymorphisms (SNPs) derived from restriction site-associated DNA sequencing (RAD-seq) and ten plastid microsatellites, and were subjected to ploidy analysis by flow cytometry. Keymore » ResultsSix major genetic groups within M. sacchariflorus were identified using SNP data: three diploid groups, comprising Yangtze (M. sacchariflorus ssp. lutarioriparius), N China and Korea/NE China/Russia; and three tetraploid groups, comprising N China/Korea/Russia, S Japan and N Japan. Miscanthus sacchariflorus ssp. lutarioriparius was derived from the N China group, with a substantial bottleneck. Japanese and mainland tetraploids originated from independent polyploidization events. Hybrids between diploid M. sacchariflorus and M. sinensis were identified in Korea, but without introgression into either parent species. In contrast, tetraploid M. sacchariflorus in southern Japan and Korea exhibited substantial hybridization and introgression with local diploid M. sinensis. ConclusionsGenetic data indicated that the land now under the Yellow Sea was a centre of diversity for M. sacchariflorus during the last glacial maximum, followed by a series of migrations as the climate became warmer and wetter. Overall, M. sacchariflorus has greater genetic diversity than M. sinensis, suggesting that breeding and selection within M. sacchariflorus will be important for the development of improved M. × giganteus. Ornamental M. sacchariflorus genotypes in Europe and North America represent a very narrow portion of the species’ genetic diversity, and thus do not well represent the species as a whole.« less